Methods of nucleic acid sample preparation for immune repertoire sequencing

Abstract

Aspects of the technology disclosed herein relate to methods of preparing and analyzing nucleic acids, e.g., nucleic acids encoding immune receptors and immunoglobulins. In some embodiments, methods for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing) are provided herein.

What is claimed is: 
     
       1. A method of preparing nucleic acids for analysis, the method comprising:
 (a) contacting a nucleic acid molecule comprising a target nucleotide sequence with a capture moiety modified primer that specifically anneals to the target nucleotide sequence under hybridization conditions; 
 (b) conducting a first strand synthesis reaction that is primed by a hybridized capture moiety modified primer and that uses the nucleic acid molecule as a template; 
 (c) conducting a second strand synthesis reaction that uses a product of the first strand synthesis reaction as a template to generate a double-stranded nucleic acid comprising a capture moiety; 
 (d) ligating an adapter nucleic acid to the double-stranded nucleic acid to produce a ligation product comprising the capture moiety; 
 (e) capturing the ligation product by contacting the ligation product with a binding partner of the capture moiety; and 
 (f) amplifying the captured ligation product by polymerase chain reaction using a target-specific primer that comprises a 3′ portion that specifically anneals to the target nucleotide sequence and a first adapter primer that specifically anneals to a complementary sequence of the adapter nucleic acid, wherein the target-specific primer comprises a 5′ tail portion that does not specifically anneal to the target nucleotide sequence. 
 
     
     
       2. The method of  claim 1 , further comprising:
 (g) amplifying an amplification product of step (f) by polymerase chain reaction using a tail primer that comprises a 3′ portion that specifically anneals to a complementary sequence of the 5′ tail portion of the target-specific primer and a second adapter primer that specifically anneals to a complementary sequence of the adapter nucleic acid, wherein the tail primer comprises a 5′ portion that does not specifically anneal to a complementary sequence of the target-specific primer. 
 
     
     
       3. The method of  claim 1 , wherein:
 step (d) comprises combining the adapter nucleic acid, the double-stranded nucleic acid, and a ligase under conditions in which the ligase ligates the adapter nucleic acid to the double-stranded nucleic acid, wherein the adapter nucleic acid that is combined with the double-stranded nucleic acid comprises a duplex portion and an overhang sequence, wherein the overhang sequence comprises a nucleotide sequence that is complementary to an overhang sequence at the 3′ end of the double stranded nucleic acid; or 
 step (d) comprises combining the adapter nucleic acid, the double-stranded nucleic acid, and a ligase under conditions in which the ligase ligates the adapter nucleic acid to the double-stranded nucleic acid, wherein the adapter nucleic acid that is combined with the double-stranded nucleic acid is single-stranded. 
 
     
     
       4. The method of  claim 1 , wherein the capture moiety modified primer comprises at least one capture moiety modified nucleotide. 
     
     
       5. The method of  claim 1 , wherein the capture moiety is a biotin moiety. 
     
     
       6. The method of  claim 1 , wherein the binding partner is streptavidin. 
     
     
       7. The method of  claim 1 , wherein, in step (d), the double-stranded nucleic acid is ligated to the adapter nucleic acid in the presence of a crowding agent. 
     
     
       8. The method of  claim 1 , wherein:
 the second strand synthesis reaction is primed by a fragment of the nucleic acid molecule hybridized to the product of the first strand synthesis reaction; or 
 the second strand synthesis is randomly primed using a plurality of random primers. 
 
     
     
       9. The method of  claim 1 , wherein the nucleic acid molecule comprises mRNA. 
     
     
       10. The method of  claim 1 , wherein the nucleic acid molecule is obtained from a sample comprising a T cell, a B cell, a leukocyte, or a mixture thereof. 
     
     
       11. The method of  claim 10 , wherein:
 the sample is obtained from a subject having, or suspected of having, a T cell malignancy or a B cell malignancy; or 
 the sample is obtained from a subject that has undergone or will undergo transplantation; or 
 the sample is obtained from a subject whose immune response to a treatment is being evaluated; or 
 the sample is obtained from a subject having, or suspected of having, a white blood cell malignancy. 
 
     
     
       12. The method of  claim 11 , wherein the subject is a human or a chordate. 
     
     
       13. The method of  claim 1 , wherein the target nucleotide sequence comprises a nucleotide sequence corresponding to a portion of a T cell receptor (TCR) gene or a B cell receptor (BCR) gene. 
     
     
       14. The method of  claim 1 , wherein the capture moiety modified primer comprises a nucleotide sequence that is complementary to an immune receptor gene or an immunoglobulin gene. 
     
     
       15. The method of  claim 14 , wherein:
 the target-specific primer specifically anneals to a constant region or a J-segment that is downstream of a CDR3; or 
 the target-specific primer specifically anneals to an exon-exon junction formed between a constant region and a J-segment, and wherein the exon-exon junction is downstream of a CDR3. 
 
     
     
       16. The method of  claim 2 , wherein:
 the first adapter primer and the second adapter primer are the same; or 
 the first adapter primer and the second adapter primer are different; or 
 the second adapter primer is nested relative to the first adapter primer. 
 
     
     
       17. The method of  claim 2 , wherein the 5′ portion of the tail primer comprises at least one of a sample index region, a molecular barcode region, and a sequencing primer site region. 
     
     
       18. The method of  claim 5 , wherein the biotin moiety comprises biotin-triethylene glycol, bis-biotin, photocleavable biotin, desthiobiotin, desthiobiotin-triethylene glycol, or biotin azide. 
     
     
       19. The method of  claim 6 , wherein the streptavidin is attached to a substrate. 
     
     
       20. The method of  claim 19 , wherein the substrate comprises a solid surface. 
     
     
       21. The method of  claim 20 , wherein the solid surface comprises a paramagnetic bead. 
     
     
       22. The method of  claim 8 , wherein the plurality of random primers are between 6 bases in length and 15 bases in length. 
     
     
       23. The method of  claim 11 , wherein the T cell malignancy or the B cell malignancy is selected from the group consisting of lymphoma, multiple myeloma, acute lymphoblastic leukemia, and chronic lymphocytic leukemia. 
     
     
       24. The method of  claim 14 , wherein the capture moiety modified primer specifically anneals to a constant region that is downstream of a complementarity determining region 3 (CDR3).